Mutagenesis of nucleic acids has been used to identify essential domains and to create nucleic acids or proteins with altered activity. A number of techniques have been used to examine the effect of simple substitution, deletion or insertion in the sequence of a nucleic acid or protein.: making processive deletions of DNA using exonuclease digestions (see, e.g., Clark & Henikoff, "Ordered Deletions Using Exonuclease III," chapter 12, Methods in Molecular Biology, volume 57, In Vitro Mutagenesis Protocols (Trower, ed., 1996)); constructing linker scanner mutations with oligonucleotides to examine promoter function or to systematically alter amino acids to examine protein function (McKnight & Kingsbury, Science 217:316-324 (1982)); constructing linker scanner mutations with PCR (Li & Shapiro, Nuc. Acids Res. 21:3745 (1993); Harlow et al., "Construction of Linker-Scanning Mutations Using PCR," chapter 26, Methods in Molecular Biology, volume 57, In Vitro Mutagenesis Protocols (Trower, ed., 1996); and Barik, "Site-directed Mutagenesis by Double Polymerase Chain Reaction," chapter 28, Methods in Molecular Biology, volume 15, PCR Protocols (White, ed., 1993)); alanine scanning mutagenesis (Cunningham & Wells, Science 44:1081 (1989)); by transposon insertion (Rass et al., Gene 130:23 (1993); Kahrs et al., Gene 167:53 (1995) and by recombination (Gray et al., J. Bacteriol. 166:635 (1986).
In addition, nucleic acid and protein function have been studied by examining the effect of chimeric molecules, e.g., by inserting mutated or shuffled nucleic acids into a gene. Typically, the mutated or shuffled domain is from the gene that is being studied, or a close homolog. For example, chimeric molecules have been made by homolog scanning (Cunningham et al., Science 241:1330 (1989)); and by PCR ("Construction and Screening of Antibody Display Libraries," Chapter 6, Phage Display of Peptides and Proteins (1996)). These techniques are used to systematically reassemble a gene encoding a protein with mutated, homologous, or shuffled domains.
Finally, techniques are also known for inserting fixed heterologous sequences into genes at known positions to create rationally designed chimeras. Examples of this latter approach include manipulation of genes such as env, adenovirus fiber protein, and retrovirus integrase to contain a heterologous, rationally designed sequences of fixed length and at specific positions (Kasahara et al., Science 266:1373 (1994); Han et al., PNAS 92:9747 (1995); Somia et al., PNAS 92:7570 (1995); Krasnykh et al., J. Virol . 72:1844 (1998); and Dildine et al., J. Virol. 72:4287 (1998)).
In order to screen large numbers of sequences and many combinations of genes, there is a need to develop novel and alternative methods of mutagenizing complex nucleic acids.